We have developed a protocol for rapid production of recombinant hemagglutinin (rHA) of influenza viruses. This procedure does not require access to virus strains. Using only DNA sequence data from the CDC/WHO influenza virus database, we chemically synthesized HA genes de novo, using a procedure optimized for protein expression in E. coli. Our protocol reduces the time required to produce an influenza vaccine to about one month compared to the 6-8 months needed for the traditional egg-based preparation. In addition to our de novo gene protocol, we have developed a cloning protocol utilizing influenza virus RNAs as templates for reverse transcription-polymerase chain reaction (rPCR) gene amplification. Using these procedures, bacterial seed clones/cultures have been constructed and used to produce pilot plant quantities of rHA. We have now developed standardized downstream processing protocols for protein extraction, purification, refolding, and vaccine formulation. A second protocol designed to broaden the protective immune response has been developed using the chemically synthesized polypeptides conjugated to genetically inactivated diphtheria toxin. Using our production protocols, time-consuming delays required by restrictive regulations related to select agents, USDA control measures for avian derived pathogens, and high-level biosafety containment are completely avoided. As shown below, we have successfully produced rHA immunogens representing five influenza A virus strains and a M2e-diphtheria toxoid conjugate vaccine: -------- H5N1 rHA Vietnam 1203/2004 Clade 1 60,000 MWt, 526 AA*, 6.9 pI, D17-Q531 HA residues, AY818135 accession number# H5N1 rHA Indonesia 5/2005 Clade 2a 59,790 MWt, 526 AA, 6.7 pI, D17-Q531 HA residues, ABP51969 accession number H1N1 rHA California 05/2009 pandemic 57,499 MWt, 512 AA, 6.9 pI, D18-Q528 HA residues, FJ966952 accession number H3N2 rHA California 7/2004 seasonal 58,561 MWt, 523 AA, 8.2 pI, Q17-K538 HA residues, 119760 accession number H5N1 rHA BHG Qinghai 1/2004 Clade 2b 60,104 MWt, 526 AA, 6.8 pI, D17-Q537 HA residues, DQ659327 assession number all A virus strains M2e peptide 2658 MWt, 22 AA, 3.9 pI, S2-D23 HA residues, various accession numbers * Amino acid residues. Numbers correspond to the full-length influenza HA0 protein before cleavage of the signal peptide and at the HA1/HA2 polybasic amino acid proteinase cleavage site. # Primary structure, i.e. amino acid residues representing the first amino acid of the mature influenza HA protein to the truncated -COOH end. Numbering is based on full length HA0 protein as recorded for the amino acid sequence assignment from the appropriate Gene Bank accession number. -------- The recombinant protein constructs were designed to represent the mature configuration of HA with the transmembrane domain, which spans the viral capsid, deleted from the carboxyl terminal. This domain was replaced with a Gly3X-His6X tag to facilitate purification of the expressed protein using Ni-ion chelating chromatography. Using five representative virus strains, rHA was produced in E. coli bacteria. The expressed protein was purified from inclusion bodies by urea solubilization and Ni-ion column chromatography. Solubilized rHA was further processed by rapid dilution into refolding buffer, extensive dialysis, and spin-filter concentration. Vaccine candidates were formulated by either adsorbing the rHA onto alum, treating the protein with formalin, or both. The purified rHA derived from the A/Vietnam/1203/2004 (H5N1), A/Indonesia/5/2005 (H5N1), and A/swine pandemic/2009 (H1N1) influenza viruses were used in vaccine formulation and immunization studies. Injected into young outbred mice three times 2 weeks apart (2.5-5 mcg/mouse), rHA induced antibodies with hemagglutination inhibition titers of 40 or higher, suggesting that rHA could induce protective immune responses against influenza virus infection (FDA guidelines require a minimal titer of 40). Our preliminary data suggest that the alum-absorbed rHA vaccine, produced in just four weeks, can fulfill the FDA requirements. Antibody neutralization experiments based on plaque reduction analysis and inhibition of viral replication within a unique tissue culture cell line are planned. Recent literature reports suggest that antibodies to the exposed N-terminal 23 amino acids (M2e) of the mature matrix 2 protein (M2) may ameliorate disease symptoms. The M2 protein forms a homotetramer hydrogen ion-channel through the viral membrane and is recognized as a target for prophylaxis and treatment with the antiviral drug Amantadine. Unlike the virion surface proteins HA and NA, which are subject to constant genetic drift and shift, the M2 protein is highly conserved. This is likely due to its protected location within the viral membrane, preventing a host immune response. However, recent studies show that when the exposed 23-amino acid M2e peptide was genetically fused to the N-terminus of hepatitis B virus core particles as a carrier, the chimeric protein conferred complete protection against a lethal heterologous influenza virus challenge in a mouse model. We linked covalently synthetic M2e peptides to a genetically detoxified diphtheria toxin (DT-H21G) via thioether linkages. MADLI-MS analyses showed an average of 7 chains of M2e per DT molecule with a mass ratio of DT: M2e = 1:0.3. This conjugate, in aqueous form or alum adsorbed, was injected s.c. into mice at 2.5 g peptide in 100 l PBS either 2 or 3 times, 2 weeks apart. Sera were collected 1 week after the last injection. High anti-M2e IgG ELISA levels were achieved after the third injection with or without alum. Both the M2e-DT conjugate and the alum absorbed M2e-DT conjugate induced statically significant serum IgG immune responses after the 2nd and 3rd injection (0.4 to 1.4 p<0.0005). An antibody response was also observed against the DT(H21G) carrier protein. Our preliminary results suggest that this candidate vaccine may induce immunity against heterologous strains of influenza A virus.